Exposure of PC12 cells to ethanol results in large changes in calcium-stimulated protease activities. Since these proteases are critical modulators of both neurotransmitter release as well as cellular toxicity and death, we are exploring the hypothesis that ethanol-mediated neural toxicity may result from calcium- activated protease dysregulation. Calpastatin, an inhibitor of calpain, is an acidic, hydrophobic protein which interacts with the hydrophobic active site(s) of mu- and m-calpains. A series of post-translational modifications of calpastatin have been described which alter the binding affinity to calpains, among them, PKC-mediated phosphorylations. Using a PC12 model, we are examining the effects of ethanol exposure and withdrawal on protein-protein interactions. Because of the hydrophobic nature of calpastatin-calpain interactions, we examined the possibility that ethanol might modify protease-calpastatin (inhibitor) complex stability. We found that exposure of PC12 cells to ethanol results in an increase in the molecular weight of calpain and calpastatin-containing protein complexes, and that this is associated with a change in protease activity. We have extended these observations by use of immunocytochemical techniques. We developed a method to quantitate alterations in spatial organization of the immunoreactive proteins of interest and applied it successfully to fluorescent immunohistochemical images. This method allows us to not only quantitate signal magnitude in situ, but also to determine if the spatial signal is altered after exposure to alcohol. We found that signal magnitudes associated with mu-calpain and calpastatin are altered by alcohol exposure, but that in the case of mu-calpain, the texture of the signal is also altered. This finding suggests that in vivo, exposure of PC12 cells to alcohol may alter calpain epitope recognition by either a protein-protein interaction, or by a post-translational modification. We are now in the process of extending these findings, and developing the capability of filming the time course and translocation of the proteins of interest in live cells immediately after exposure to alcohol. This will increase our understanding of the mechanisms involved in the regulation of calpain activity by alcohol.